Major neutralization site of hepatitis e virus and use of this neutralization site in methods of vaccination and in methods of screening for neutralizing antibodies to hepatitis e virus

ABSTRACT

The invention describes the identification of major neutralization site of hepatitis E virus (HEV) and the use of this neutralization site in methods of vaccination and in methods of screening for neutralizing antibodies to HEV. The invention also describes the isolation and characterization of neutralizing chimpanzee monoclonal antibodies reactive to the neutralization site and the use of these antibodies in the diagnosis, treatment and prevention of HEV.

FIELD OF THE INVENTION

[0001] The present invention relates to the identification of a majorneutralization site of hepatitis E virus (HEV) and the use of thisneutralization site in methods of vaccination and in methods ofscreening for neutralizing antibodies to HEV. The invention furtherrelates to the isolation and characterization of neutralizing antibodiesreactive to the neutralization epitope and the use of these antibodiesin the diagnosis, treatment and prevention of HEV.

BACKGROUND OF THE INVENTION

[0002] Hepatitis E is endemic in many countries throughout thedeveloping world, in particular on the continents of Africa and Asia.The disease generally affects young adults and has a very high mortalityrate, up to 20%, in pregnant women (Mast, 1993; Tsega, 1992; Khuroo,1981). The causative agent, hepatitis E virus (HEV), is transmittedprimarily by the fecal-oral route, often through contaminated water(Purcell, 1996)). The availability of sensitive serological tests forHEV has permitted detailed assessment of the prevalence of HEVinfection. In regions where HEV is endemic, anti-HEV antibodies havebeen detected in sera from convalescent individuals as well as from thegeneral population. Surprisingly, in industrialized countries, such asthe United States, where hepatitis E is not endemic, a significantproportion of healthy individuals within the general population areseropositive (up to 20% in some areas (Thomas, 1997; Mast, 1997)).However, clinical hepatitis E is rare in these countries and individualsusually acquire their infection during travel to a region that isendemic or epidemic for HEV.

[0003] It has been suggested that animals serve as reservoirs for HEV insome regions, and human infections may, in part, be zoonoses. There havebeen several reports of HEV-specific antibody (anti-HEV) in animals(Clayson, 1995; Karetnyi, 1993; Arankalle, 1994; Kabrane-Lazizi, 1999).Furthermore, an HEV-like virus was recently isolated from naturallyinfected swine in the United States (Meng, 1997). The four genotypes ofHEV identified based on nucleotide sequence diversity are Asian/African,Mexican, U.S. and the New Chinese. To-date, only one serotype of HEV hasbeen found. Therefore, it may be possible to produce a broadlyprotective vaccine in the near future.

[0004] Studies have shown that passively transferred anti-HEVsignificantly reduced virus shedding in feces, and abrogated disease innon-human primates challenged with a high dose of HEV (Tsarev, 1994).The findings suggest that immunoglobulin preparations, similar to thoseused for protection against hepatitis A, would be efficacious againsthepatitis E. Field studies in India performed using pools of normalserum immunoglobulin collected from HEV endemic regions did not showprotection from HEV infection or disease (Joshi, 1985; Khuroo, 1992;Zhuang, 1991). It is likely that the titer of anti-HEV antibodies inthose studies was too low to have a protective effect. As pooled normalhuman serum is unlikely to be useful as an immunoprophylactic reagentagainst HEV, neutralizing monoclonal antibodies to HEV could be used toproduce a high titer immunoglobulin preparation which might protectagainst hepatitis E virus.

[0005] Antibody phage display libraries provide a powerful tool for theisolation of human antibodies to important viral pathogens. Antibodyphage display libraries are constructed from variable heavy and lightchain antibody genes using a phage display vector specifically designedfor the expression of antibody fragments to an antigen (Winter, 1994; deKruif, 1996; Burton, 1994). From such libraries, large numbers of humanmonoclonal antibodies to an antigen of choice can be cloned andisolated. The technique provides new opportunities to produce highaffinity human monoclonal antibodies for use in passiveimmunoprophylaxis. To date, monoclonal antibodies to a number of viralantigens, for example, human immunodeficiency virus-1 gp120 (Thompson,1996; Geoffroy, 1994; Burton, 1991; Ditzel, 1997), measles virus(Bender, 1994), and respiratory syncitial virus F protein (Crowe, 1994),have been isolated.

[0006] The identification of neutralization epitopes of HEV provides analternative method for the production of neutralizing antibodies to HEV.

SUMMARY OF THE INVENTION

[0007] The present invention relates to the identification of aneutralization site of hepatitis E virus (HEV) which consists of one ormore neutralization epitopes of HEV. The neutralization site is apolypeptide about 30 amino acids in length spanning from amino acids 578to 607 of the ORF2 gene (capsid gene) of HEV. The neutralization site isconserved among genetically divergent HEV strains.

[0008] The invention also relates to the use of the neutralization siteor the epitope(s) contained within the neutralization site as animmunogen to elicit the production in mammals of antibodies that caneffectively neutralize one or more strains of HEV.

[0009] The invention also relates to the use of the neutralization siteor the epitope(s) contained within the neutralization site as vaccine toeffectively prevent, and/or reduce the incidence of HEV infection. Anepitope or antigenic determinant is typically about six amino acidresidues.

[0010] The invention also relates to pharmaceutical compositionscomprising the neutralization site or the epitope(s) contained withinthe neutralization site.

[0011] The invention further relates to methods of producingneutralizing antibodies to HEV comprising administering thepharmaceutical compositions of the invention to a mammal in an amounteffective to stimulate the production of neutralizing antibodies to HEV.

[0012] The present invention also relates to the isolation andcharacterization of two neutralizing chimpanzee monoclonal antibodieswhich are reactive with the neutralization site or the epitope(s)contained within the neutralization site of the invention. Thesemonoclonal antibodies react with genetically divergent HEV strains.

[0013] The invention also relates to the heavy and light chainimmunoglobulin variable region amino acid sequences of theseneutralizing monoclonal antibodies to HEV, and to the nucleic acidmolecules encoding the amino acid sequences.

[0014] The present invention also relates to the use of the neutralizingmonoclonal antibodies of the invention in the detection of HEV infectionin animals, especially mammals, and most especially humans.

[0015] The neutralizing monoclonal antibodies of the present inventionare particularly advantageous for use in the development ofprophylactic, therapeutic and diagnostic agents for the prevention andtreatment of hepatitis E and detection of human HEV.

[0016] The invention therefore also relates to pharmaceuticalcompositions which comprise the neutralizing antibodies of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIG. 1(a) shows restriction fragment analysis of sevenHEV-specific Fab clones. Plasmid DNA was digested with Bst N1 and analiquot was electrophoresed on a 3% agarose gel. FIG. 1(b) shows theamino acid sequence of the γ1-chains of monoclonal antibodies HEV#4 and#31.

[0018]FIG. 2 shows a Western blot of HEV ORF2 protein (55 kD)immunoblotted with chimpanzee 1441 post-immune serum (lane A), HEV#4(lane B), and HEV#31 (lane C), respectively. The positions of molecularweight markers are shown on the left side of the blot.

[0019]FIG. 3 shows the result of a radioimmunoprecipitation assay usingmonoclonal antibodies HEV#4 and #31. FIG. 3(a) shows a schematicrepresentation of the ORF2 proteins truncated at the C-terminus. FIG.3(b) shows an autoradiograph of the radioimmunoprecipitation assay usinga pool of the six ³⁵S-labeled ORF2 truncation products.

[0020]FIG. 4 shows an ELISA assay testing the crossreactivity of HEV#4and HEV#31 with recombinant baculovirus-expressed ORF2 protein from thePakistani (SAR-55) and swine HEV strains.

[0021]FIG. 5 shows in vitro neutralization of HEV with in vivomonitoring in rhesus monkeys inoculated with HEV treated with (a)chimpanzee 5835 pre-immune serum, (b) HBV#8 Fab, (c) HEV#4, (d) HEV#31,and (e) chimpanzee 5835 hyper-immune serum.

[0022]FIG. 6 shows a comparison of amino acid residues 578 to 607 of theORF2 protein from different HEV strains.

[0023]FIG. 7(a) shows the heavy and light chain immunoglobulin variableregion amino acid sequences of HEV#4 and the nucleotide sequencesencoding the amino acid sequences. FIG. 7(b) shows the heavy and lightchain immunoglobulin variable region amino acid sequences of HEV#31 andthe nucleotide sequences encoding the amino acid sequences.

[0024]FIG. 8 shows the CDR3 sequences of the γ1-heavy chain of 17monoclonal antibodies including HEV#4 and HEV#31 isolated from the phagedisplay library.

[0025]FIG. 9 shows in vitro neutralization of HEV with in vivomonitoring in rhesus monkeys inoculated with HEV (SAR-55 strain) treatedwith (a) EBL#2 Fab, (b) EBL#89 Fab, and (c) chimpanzee Ch5835hyper-immune serum.

[0026]FIG. 10 shows the topography of the epitopes recognized by theantibodies listed in FIG. 8. Where two circles overlap there is greaterthan 50% inhibition of binding between the antibody pair.

[0027]FIG. 11 shows a topographical map of the HEV ORF2 antigenic siteoverlaid with epitope recognition data from radioimmunoprecipitationstudies. The locations of the neutralization and non-neutralizationepitopes on the topographical map of the HEV ORF2 antigenic site areindicated with a “✓” and a “X”, respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention relates to a peptide of at least 30 aminoacids in length, spanning amino acids 578 to 607 of the open-readingframe 2 gene (capsid gene) of hepatitis E virus (HEV), which has beenidentified as a neutralization site of the virus.

[0029] The results described herein show that the peptide comprisingamino acid 112 to amino acid 578 of the open-reading frame 2 genereacted weakly with the neutralizing antibodies to HEV compared to thepeptide comprising amino acid 112 to amino acid 607 which reactedstrongly with the neutralizing antibodies to HEV. As the negative resultobtained with the peptide comprising amino acid 112 to amino acid 578may be due to a disruption of a neutralization epitope at the aminoterminus of the 578-607 sequence, it is understood that the polypeptideof the invention may extend 5-10 amino acids amino-terminal to aminoacid 578 such that it encompasses from about amino acid 572, or 573 toabout amino acid 607, more preferably, from about amino acid 568 toabout amino acid 607 of the open-reading frame 2 gene.

[0030] It is further understood that the neutralization site consists ofone or more neutralization epitopes of HEV. The nature and the locationof the neutralization epitope(s) within the neutralization site can bedetermined by deletional or mutational analyses described herein. Aneutralization epitope is understood to be composed of at least 6 aminoacids, preferably 6 to 8 amino acids.

[0031] As the neutralization site is conserved among geneticallydivergent HEV strains, it is understood that although the neutralizationsite of the invention was identified as a polypeptide about 30 aminoacids in length spanning from amino acids 578 to 607 of the ORF2 gene ofHEV strain SAR-55, the invention also encompasses a neutralization siteand epitope(s) from corresponding regions of the ORF2 gene of other HEVstrains.

[0032] It is further understood that substitution of amino acidresidue(s) within the neutralization site or neutralization epitope(s)of the invention may result in polypeptides which have similarneutralization properties as the neutralization site or theneutralization epitope(s) set forth above, and which are capable ofdirecting the production of antibodies that are reactive with theneutralization site or epitope(s) of the invention described above. Itshould be noted that the neutralization site set forth above representsa preferred embodiment of the present invention.

[0033] Deletional or mutational studies of the neutralization site ofthe invention will allow the engineering of broadly reactiveneutralization epitopes of HEV. Such studies will also allow theengineering of genotype-specific epitopes of HEV which are useful asdiagnostic agents for various genotypes of HEV.

[0034] The invention also relates to the use of the neutralization siteor the neutralization epitope(s) of the invention as an immunogen toelicit the production in mammals of antibodies that can effectivelyneutralize one or more strains of HEV.

[0035] The term “antibodies” is used herein to refer to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules. Exemplary antibody molecules are intact immunoglobulinmolecules, substantially intact immunoglobulin molecules and portions ofan immunoglobulin molecule, including those portions known in the art asFab, Fab′, F(ab′)₂, F_(d) and F(v) as well as chimeric antibodymolecules.

[0036] In one embodiment, the neutralizing antibodies are produced byimmunizing a mammal with a peptide or peptide fragments encoding theneutralization site or the neutralization epitope(s) of the invention.In another embodiment, the neutralizing antibodies are produced byimmunizing a mammal with nucleic acids encoding the neutralization siteor the neutralization epitope(s) of the invention. In yet anotherembodiment, the neutralizing antibodies are produced by immunizing amammal with peptides bridging the ORF2 region of HEV. The antibodymolecules may then be collected from the mammal if they are to be usedin immunoassays or for providing passive immunity.

[0037] The antibody molecules of the present invention may be polyclonalor monoclonal. Monoclonal antibodies may be produced by methods known inthe art. Portions of immunoglobulin molecules may also be produced bymethods known in the art.

[0038] The antibody of the present invention may be contained in variouscarriers or media, including blood, plasma, serum (e.g., fractionated orunfractionated serum), hybridoma supernatants and the like.Alternatively, the antibody of the present invention is isolated to theextent desired by well known techniques such as, for example, by usingDEAE SEPHADEX, or affinity chromatography. The antibodies may bepurified so as to obtain specific classes or subclasses of antibody suchas IgM, IgG, IgA, IgG₁, IgG₂, IgG₃, IgG₄ and the like. Antibodies of theIgG class are preferred for purposes of passive protection.

[0039] In addition to its use in generating neutralizing antibodies toHEV, the neutralization site or the neutralization epitope(s) of theinvention can be used as an immunogen to stimulate the production of aprotective humoral and/or cellular immune response to HEV.

[0040] In one embodiment, the immunogen may be a partially orsubstantially purified peptide or peptide fragments encoding theneutralization site or the neutralization epitope(s) of the invention.In another embodiment, the immunogen may be a construct in which thepeptide or peptide fragments of the invention is incorporated into alive virus vector, for example, a vaccinia virus or adenovirus vector,which may contain neutralization epitopes of other pathogens. In yetanother embodiment, the immunogen may be a construct in which thepeptide or peptide fragments of the invention is incorporated intoproteins such as the hepatitis B surface antigen or the hepatitis B coreantigen. In another embodiment, the immunogen may be a construct inwhich the peptide or peptide fragments of the invention is incorporatedinto a mosaic protein which contains antibody binding site(s) of otherantigens. In another embodiment, the immunogen may be a cell, celllysate from cells transfected with a recombinant expression vector, or aculture supernatant containing the peptide encoding the neutralizationsite or the neutralization epitope(s) of the invention. In anotherembodiment, the immunogen may be a DNA construct encoding theneutralization site or the neutralization epitope(s) of the invention.

[0041] While it is possible for the immunogen to be administered in apure or substantially pure form, it is preferable to present it as apharmaceutical composition, formulation or preparation. For example, theimmunogen can be used in a suitable diluent such as saline or water, orcomplete or incomplete adjuvants. In a preferred embodiment, theimmunogen is coupled to a carrier to make the peptide more immunogenic.Examples of such carrier molecules include but are not limited to bovineserum albumin (BSA), keyhole limpet hemocyanin (KLH), tetanus toxoid,and the like. The immunogen can be administered by any route appropriatefor antibody production such as intravenous, intraperitoneal,intramuscular, subcutaneous, and the like.

[0042] The effective amount of peptide per unit dose sufficient toinduce an immune response depends, among other things, on the species ofmammal inoculated, the body weight of the mammal and the choseninoculation regimen, as well as the presence or absence of an adjuvant,as is well known in the art. Inocula typically contain peptideconcentrations of about 1 microgram to about 50 milligrams perinoculation (dose), preferably about 10 micrograms to about 10milligrams per dose, most preferably about 100 micrograms to about 5milligrams per dose.

[0043] The term “unit dose” as it pertains to the inocula refers tophysically discrete units suitable as unitary dosages for mammals, eachunit containing a predetermined quantity of active material(polypeptide) calculated to produce the desired immunogenic effect inassociation with the required diluent.

[0044] The immunogen may be administered once or at periodic intervalsuntil a significant titer of anti-HEV antibody is produced. The antibodymay be detected in the serum using an immunoassay.

[0045] To monitor the antibody response of individuals administered thecompositions of the invention, antibody titers may be determined. Inmost instances it will be sufficient to assess the antibody titer inserum or plasma obtained from such an individual. Decisions as towhether to administer booster inoculations or to change the amount ofthe composition administered to the individual may be at least partiallybased on the titer.

[0046] The titer may be based on an immunobinding assay which measuresthe concentration of antibodies in the serum which bind to theneutralization site or the neutralization epitope(s) contained withinthe neutralization site of the invention. The ability to neutralize invitro and in vivo biological effects of the viruses of this inventionmay also be assessed to determine the effectiveness of the immunization.

[0047] Where immunoassays are involved, such kits may contain a solidsupport, such as a membrane (e.g., nitrocellulose), a bead, sphere, testtube, microtiter well, rod, and so forth, to which a receptor such as anantibody specific for the target molecule will bind. Such kits can alsoinclude a second receptor, such as a labeled antibody. Such kits can beused for sandwich assays. Kits for competitive assays are alsoenvisioned.

[0048] The invention also relates to the use of the immunogens of thepresent invention as vaccines for either a prophylactic or therapeuticpurpose. When provided prophylactically, a vaccine(s) of the inventionis provided in advance of any exposure to any one or more of the HEVstrains or in advance of any symptoms due to infection of the viruses.The prophylactic administration of a vaccine(s) of the invention servesto prevent or attenuate any subsequent infection of these viruses in amammal. When provided therapeutically, a vaccine(s) of the invention isprovided at (or shortly after) the onset of infection or at the onset ofany symptom of infection or any disease or deleterious effects caused bythese viruses. The therapeutic administration of the vaccine(s) servesto attenuate the infection or disease. The vaccine(s) of the presentinvention may, thus, be provided either prior to the anticipatedexposure to the viruses of this invention or after the initiation ofinfection.

[0049] The immunogens of the invention may be supplied in the form of akit, alone, or in the form of a pharmaceutical composition.

[0050] The present invention also relates to neutralizing chimpanzeemonoclonal antibodies to HEV, where the antibodies are isolated as Fabfragments from a phage display library prepared from RNA isolated frombone marrow lymphocytes of a chimpanzee experimentally infected with theHEV strain SAR-55, the hepatitis A virus (HAV), the hepatitis B virus(HBV), the hepatitis C virus (HCV), and the hepatitis D virus (HDV).

[0051] The present invention thus relates to neutralizing chimpanzeemonoclonal antibodies having specified heavy (H) and light (L) chainimmunoglobulin variable region amino acid sequences in pairs (H:L) whichconfer the ability to bind to the neutralization epitope of theinvention.

[0052] The present invention therefore relates to the heavy chainimmunoglobulin variable region amino acid sequences and the light chainimmunoglobulin variable region amino acid sequences shown in FIG. 7.

[0053] The present invention also relates to nucleic acid moleculesencoding the heavy and light chain immunoglobulin variable region aminoacid sequences of this invention where these sequences are shown in FIG.7.

[0054] Of course, due to the degeneracy of the genetic code, variationsare contemplated in the sequences shown in FIG. 7 which will result innucleic acid sequences that are capable of directing production ofantibodies that are identical to the antibodies of the invention. Itshould be noted that the DNA sequences set forth above represent apreferred embodiment of the present invention.

[0055] The invention further relates to methods of making neutralizingchimpanzee monoclonal antibodies from the phage display librarydescribed herein. In a preferred embodiment, the method for isolating aneutralizing monoclonal antibody from the phage display library involves(1) using immunoaffinity techniques such as panning to select phageparticles that immunoreact with the neutralizationepitope of theinvention; (2) infecting bacteria with the selected phage particles; (3)preparing and analyzing the phagemid DNA from the colonies recovered;and (4) expressing and purifying soluble Fab fragments from clones ofinterest for further analysis.

[0056] The invention also relates to the use of the neutralizingmonoclonal antibodies as diagnostic agents.

[0057] The antibodies can be used as an in vitro diagnostic agent totest for the presence of HEV in biological samples. In one embodiment, asample such as biological fluid or tissue obtained from an individual iscontacted with a diagnostically effective amount of one or more of thehuman monoclonal antibodies of this invention under conditions whichwill allow the formation of an immunological complex between theantibody and the HEV antigen that may be present in the sample. Theformation of an immunological complex, which indicates the presence ofHEV in the sample, is then detected by immunoassays. Such assaysinclude, but are not limited to, radioimmunoassays, Western blot assay,immunofluorescent assay, enzyme immunoassay, chemiluminescent assay,immunohistochemical assay and the like.

[0058] The invention also relates to the use of the monoclonalantibodies of the invention in passive immunoprophylaxis and passiveimmunotherapy of HEV infection.

[0059] When used in passive immunotherapy, the patient is administered atherapeutically effective amount of one or more neutralizing humanmonoclonal antibodies. The passive immunotherapy of this invention maybe practiced on individuals infected with HEV; passive immunoprophylaxismay be practiced on individuals at risk of HEV infection.

[0060] A prophylactically or therapeutically effective amount of amonoclonal antibody for individual patients may be determined bytitrating the amount of antibody given to the individual to arrive atthe therapeutic or prophylactic effect while minimizing side effects.The effective amount can be measured by serological decreases in theamount of HEV antigens in the individual. The plasma concentration forindividuals receiving the treatment is typically between 0.1 ug/ml to100 ug/ml.

[0061] The monoclonal antibodies of this invention may be administeredvia one of several routes including, but not limited to intravenous,intraperitoneal, intramuscular, subcutaneous, transdermal and the like.

[0062] The present invention therefore relates to pharmaceuticalcompositions comprising at least one antibody of the invention and apharmaceutically acceptable carrier where such carriers may includephysiologically acceptable buffers, for example, saline or phosphatebuffered saline.

[0063] The present invention further relates to anti-idiotypicantibodies to the monoclonal antibodies of this invention. In oneembodiment, an anti-idiotypic antibody can be prepared by immunizing ahost animal with a monoclonal antibody of this invention by methodsknown to those of skill in the art. To eliminate an immunogenic responseto the Fc region, antibodies produced by the same species as the hostanimal can be used or the Fc region of the administered antibodies canbe removed. The anti-idiotypic antibodies produced can be used toprepare pharmaceutical compositions rather than using the monoclonalantibodies of this invention.

[0064] The present invention includes compositions of the antibodiesdescribed above, suitable for parenteral administration including, butnot limited to, pharmaceutically acceptable sterile isotonic solutions.Such solutions include, but are not limited to, saline and phosphatebuffered saline for intravenous, intramuscular, intraperitoneal, orsubcutaneous injection, or direct injection into a joint or other area.

[0065] In providing the antibodies of the present invention to arecipient mammal, preferably a human, the dosage of administeredantibodies will vary depending upon such factors as the mammal's age,weight, height, sex, general medical condition, previous medical historyand the like.

[0066] In general, it is desirable to provide the recipient with adosage of antibodies which is in the range of from about 5 mg/kg toabout 20 mg/kg body weight of the mammal, although a lower or higherdose may be administered. In general, the antibodies will beadministered intravenously (IV) or intramuscularly (IM).

[0067] The present invention will now be described by way of examples,which are meant to illustrate, but not limit, the scope of theinvention.

EXAMPLE Materials and Methods

[0068] Donor Animal

[0069] Bone marrow was aspirated from the iliac crest of chimpanzee1441. The animal had been experimentally infected with HAV, HBV, HCV,HDV and HEV. Prior to the aspirate being taken, the animal was boostedwith the commercial HAV vaccine (HAVRIX, SmithKline Beecham), HBVvaccine (Engerix-B, SmithKline Beecham), and purifiedbaculovirus-expressed HEV ORF2 protein. The bone marrow lymphocytes wereseparated on a Ficoll gradient and stored as a viable single cellsuspension in 10% dimethyl sulfoxide, 10% fetal calf serum and RPMI 1640medium (BioWhittaker) in liquid nitrogen.

[0070] Construction of γ1/κ Antibody Phase Library

[0071] Total RNA was extracted from −10⁸ bone marrow lymphocytes (RNAIsolation Kit; Stratagene) and mRNA was reverse transcribed into cDNAusing an oligo (dT) primer (Gibco/BRL). The cDNAs were amplified by PCRusing rTth DNA polymerase (Perkin Elmer). Thirty cycles of 94° C. for 15s, 52° C. for 50 s, and 68° C. for 90 s were performed. Chimpanzeeκ-chain genes were amplified using primers specific for the humanκ-chain genes. Fd segments (variable and first constant domains) of thechimpanzee γ1-chain genes were amplified with nine family-specific humanVH primers recognizing the 5′ end of the genes [Barbas, 1991; Persson,1991] and a chimpanzee γ1-specific 3′ primer(5′-GCATGTACTAGTTGTGTCACAAGATTTGGG-3′) (3′ primer sequence determinedfrom Vijh-Warrier et al [Vijh-Warrier, 1995]).

[0072] The amplified κ-chains were cloned into the pComb3H phage displayvector as described by Williamson et al. [Williamson, 1993]. Theamplified γ1-chains were cloned into pGEM-T cloning vector (Promega) viathe additional adenosine nucleotide added by the rTth DNA polymerase atthe 3′ ends of the PCR product. The γ1-pGEM-T clones were transformedinto Escherichia coli XL-1 Blue (Stratagene) and expanded into a volumeof 2 liters by solid phase amplification as described in Glamann et al.[Glamann, 1998]. The γ1-pGEM-T library was digested with Xho I and Spe I(Boehringer Mannheim), and ligated into the κ-chain pComb3H library,also digested with Xho I and Spe I. The ligated products weretransformed into E.coli XL-1 Blue. Transformants were expanded into avolume of 2 liters by solid phase amplification. The final library of1.9×10⁷ clones was stored in 12.5% glycerol-LB broth at −80° C. untiluse.

[0073] Panning and ELISA Reagents

[0074] HEV ORF 2 proteins (55 kD) from the Pakistani strain SAR-55, andswine HEV were produced in baculovirus and purified according toRobinson et al. [Robinson, 1998]. In all panning and enzyme-linkedimmunosorbant assay (ELISA) experiments, HEV ORF 2 proteins were dilutedto 1.0 μg ml⁻¹ in 50 mM sodium carbonate buffer (pH 9.6), and adsorbedto EIA/RIA A/2 (ELISA) plates (Costar) overnight at 4° C. A goatanti-human IgG (H+L)-specific antibody (Pierce) was used to detect Fabproduction. This was coated to microtiter wells at a dilution of 1:1000,in 50 mM sodium carbonate buffer (pH 9.6), as above.

[0075] Library Screening

[0076] Screening of the combinatorial library was carried out accordingto the method described by Barbas et al. [Barbas, 1991] and Williamsonet al. [Williamson, 1993]. Approximately 10⁹ bacteria from the librarystock were inoculated into Luria-Bertani (LB) broth (Gibco/BRL)supplemented with 100 μg ml⁻¹ ampicillin and 1% (v/v) glucose (Sigma),and grown up and infected with helper phage, VCS M13 (Stratagene), at amultiplicity of infection of 50, to produce the library displayed on thesurface of phage particles. Phage were panned on HEV ORF2-coated ELISAwells; in all, four rounds of panning were performed. Afteramplification of the selected library, the phagemid DNA was extractedand the vector modified by restriction enzyme digestion to remove thebacteriophage coat protein III-encoding region of the phage [Bender,1993]. The phagemid DNAs were religated and transformed into E.coli XL-1Blue to allow soluble Fabs to be produced. Colonies were inoculated intoindividual wells of microtiter plates and grown in LB broth at 30° C.overnight. Fab production was induced according to Glamann et al.[Glamann, 1998], and the bacterial supernatants tested by ELISA forreactivity with HEV ORF2 and for the presence of Fab.

[0077] Fab Production and Purification

[0078] Fab purification was facilitated by modification of the vector,pComb3H, to encode a six-histidine tail at the end of the soluble Fabfragment (modification carried out by, and detailed in Glamann et al.[Glamann, 1998]). Bacterial culture and Fab fragment purification werecarried out as described by Glamann et al. [Glamann, 1998]. Proteinconcentrations were determined by both dye binding assay (Bio-Rad) andA_(280nm) (using the extinction coefficient of 1.4 optical density unitsequivalent to 1.0 mg ml⁻¹). The Fab purity was determined bypolyacrylamide gel electrophoresis with colloidal Coomassie bluestaining (Sigma).

[0079] ELISA Analysis of Fab Reactivity and Cross-reactivity

[0080] Protein antigens were coated onto ELISA microtiter plates at 1.0μg ml⁻¹ (HEV ORF2 protein) or 10.0 μg ml⁻¹ (thyroglobulin, lysozyme, andcytochrome C (Sigma)). Antigen-coated wells were blocked for 1 h at roomtemperature with 3% bovine serum albumin (BSA)-PBS, washed twice withPBS-Tween 20 (0.05 % (v:v)), and 50 μl of crude or purified Fab wasadded to the wells. After 1 h incubation at 37° C., the plates werewashed six times with PBS-Tween 20. Bound Fab were detected with 1:1500dilution of a goat anti-human F(ab′)₂ alkaline phosphatase labeledsecondary antibody (Pierce). The assay color was developed using 1 mgml⁻¹ p-nitrophenyl phosphate (Sigma) in diethanolamine buffer (Pierce).Optical density was determined at 405 nm with a reference wavelength of650 nm.

[0081] Nucleic Acid Sequencing, Analysis and Bst N1 Fingerprinting ofHEV-specific Fab Clones

[0082] Nucleic acid sequencing was performed with the ABI PRISM DyeTerminator Cycle Sequencing Ready Reaction kit by using Ampli-Taq DNAPolymerase (Perkin-Elmer) and the following sequencing primers: heavychain, 5′-ATTGCCTAC-GGCAGCCGCTGG-3′(HC1) and5′-GGAAGTAGTCCTTGACCAGGC-3′(HC4); κ chain,5′-ACAGCTATCGCGATTGCAGTG-3′(LC1) and 5′-CACCTGATCCTCA-GATGGCGG-3′(LC4)[Glamann, 1998]. The resulting sequences were analyzed using GeneWorks(Oxford Molecular Group) software package. Sequence similarity searcheswere performed using the V-BASE program, which is a compilation of allthe available human variable segment Ig germ line sequences [Cook,1995]. For Bst N1 (New England Biologicals) fingerprinting, onemicrogram of plasmid DNA was digested with 1 U of enzyme overnight at60° C. The restriction patterns were analyzed on a 3% agarose gel.

[0083] Western Blotting

[0084] HEV ORF2 protein was heated in 2× Laemelli buffer [Laemelli,1970] and run in a single well 10% polyacrylamide gel (Novex).Electrophoretic transfer of the protein to a nitrocellulose membrane wascarried out at 250 mA for 1 h at 4° C. The membrane was blocked for 30min with 5% skimmed milk in PBS prior to overnight incubation with equalconcentrations of purified of HEV#4 and HEV#31, or a 1:100 dilution ofchimpanzee 1441 serum, at 4° C. After 6 of 10 min washes each,anti-human IgG (Fab-specific) alkaline phosphatase-labeled (Pierce)secondary antibody was added at a dilution of 1:5000 in 5% skimmedmilk-PBS. After 1 h, the blot was washed, and NBT/BCIP substrate(Pierce) added.

[0085] Affinity Determinations Using BIAcore™

[0086] The gold-coated sensor chips, CM-5, are coated with acarboxylated dextran polymer matrix to which the HEV ORF2 protein wasamine coupled (Pharmacia Biosensor). The carboxyl groups on the dextransurface were activated with 35 μl of a 50:50 (v/v) solution ofN-hydroxylsuccinamide (NHS) and N-ethyl-N′-(3-diethylaminopropyl)carbodiimide (EDC). HEV ORF2 protein was diluted in 10 mM sodium acetate(pH 4.5) prior to coupling. After washing the sensor surface with HBS[10 mM HEPES (pH 7.5), 0.15 M NaCl, 3.4 mM EDTA, 0.05% Tween 20], theremaining active binding sites on the chip were blocked by the additionof ethanolamine hydrochloride.

[0087] Affinity measurements were made using three different coatingconcentrations of HEV ORF2 protein, one for each flow cell on the chip,with the fourth flow cell left uncoated and blocked as a control. Theaffinity measurements were initiated by passing HBS over the sensorsurface for 100 s at 10 μl min⁻¹ then 50 μl of Fab was injected at thesame flow rate. The kinetic analysis was performed twice. The first chipwas coated with 37 Resonance Units (RU), 203 RU and 334 RU of HEV ORF2protein. The second chip was coated with 363 RU, 225 RU and 106 RU ofHEV ORF2 protein Serial dilutions of HEV#4 and HEV#31 were made in HBSbuffer. Eight dilutions of each monoclonal antibody were tested over thesensor surfaces; the HEV#4 dilutions ranged from 0.5 to 200 nM, andHEV#31 from 1.0 to 400 nM. Between each monoclonal antibody bindingphase the sensor surface was regenerated with a one minute pulse ofregeneration buffer [1 M NaCl, 50 mM NaOH]. The level of monoclonalantibody binding to the sensor surface was reproducible followingregeneration.

[0088] Fab Biotinylation and Indirect Competition ELISA

[0089] Prior to biotinylation, purified HEV#4 and HEV#31 were dialyzedagainst PBS overnight at 4° C. Conjugation with biotin was carried outas per the manufacturer's instructions (Pierce). The biotinylated Fabswere titrated on HEV ORF2-coated wells to determine a dilution that wassub-saturating and gave an O.D. reading of approximately 1.0 atA_(405nm). For the competition assay, three-fold dilutions of unlabeledand unpurified Fab were incubated on HEV ORF2-coated wells for 1 h at37° C., then washed four times with PBS-Tween 20. A single dilution ofbiotinylated Fab was added to the wells and incubated for 1 h at 37° C.After four washes with PBS-Tween 20, strepavidin-alkaline phosphatase(Pierce) was added at a 1:500 dilution and incubated for 1 h at 37° C.The color was developed as described above.

[0090] In Vitro Neutralization of HEV with in Vivo Monitoring in RhesusMonkeys

[0091] Rhesus monkeys that were anti-HEV negative (<1:100) in asensitive ELISA [Tsarev, 1993 #171] were used in this study. Ten monkeyswere divided into five groups of two animals each. A 10% stoolsuspension of the Pakistani HEV strain, SAR-55, was diluted such thateach animal would receive 64 monkey infectious doses 50 (MID₅₀). Thiswas incubated with either purified Fab or chimpanzee serum. HEV#4,HEV#31 and an irrelevant Fab, HBV#8, were diluted to 1.9 mg ml⁻¹ in 10%BSA/PBS. Ten percent solutions of chimpanzee 5835 pre-immune serum andhyperimmune serum were made in 10% BSA/PBS. Virus and antibody weremixed and incubated for 1 h at room temperature, then at 4° C.overnight. The inoculum was divided in half and diluted with 1 ml ofice-cold PBS prior to intravenous inoculation. Serum samples werecollected prior to inoculation and for 20 weeks thereafter. Sera wereassayed for levels of alanine amino transferase (ALT) with commerciallyavailable tests (Metpath Inc.). The anti-HEV ELISA was performed asdescribed elsewhere [Tsarev, 1993]. Seroconversion to HEV was used asthe criterion for infection.

[0092] Construction of Clones Expressing Truncated SAR-55 ORF2 Proteins

[0093] Truncated SAR-55 ORF2 proteins were made by PCR amplification ofportions of the HEV ORF2 gene from pHEVORF2 63.2 [Tsarev, 1993].Truncations from the carboxy-terminal end of the protein were made usingthe following primers: 5′SAR-55 aa112(5′-ATGGCGGTCGCTCCGGCCCATGACACCC-3′), with one of 3′SAR-55 aa208(5′-CTATTAAATGGAGATAGCGTAGCCACCAACAGC-3′), 3′ SAR-55 aa308(5′-CTATTAGCGGAACTCAAGTTCGAGGGCAAAGTC-3′), 3′ SAR-55 aa408(5′-CTATTAAGTCGGCTCGCCATTGGCTGAGACGAC-3′), 3′ SAR-55 aa508(5′-CTATTACTGCGCGCCGGTCGCAACATTAACCAA-3′), 3′ SAR-55 aa578(5′-CTATTACCGATGCCCAGCGGCATTCTCAACG-3′), or 3′ SAR-55 aa607(5′-CTATTATAGCACAGAGTGGGGGGCTAAAACA-3′). Amino acids 112 through 607comprise the 55 kD protein used previously in vaccination studies[Tsarev, 1994; Tsarev, 1997]. Amino acids 112 through 578 represent a 53kD protein, which readily formed virus-like particles, when examinedunder the electron microscope. The 5′ SAR-55 aa112 primer wasphosphorylated with T4 polynucleotide kinase prior to PCR amplification,and the products were cloned into the mammalian expression vector,pCR3.1 (Unidirectional TA cloning kit, Invitrogen).

[0094] In Vitro Transcription and Translation, andRadioimmunoprecipitation of ³⁵S-labeled Truncated SAR-55 ORF2 Proteins

[0095] In vitro transcription and translation of the truncated SAR-55ORF2 clones aa208 to aa607 were carried out according to themanufacturer's protocol (T7 TNT in vitro transcription/translation,Promega) using ³⁵S-methionine (Redivue, Amersham) as the radiolabel. Thesix truncated ORF2 products were visualized on a 10-20% PAGE gelfollowed by autoradiography, then pooled. Five microliters of pooledtruncated products were mixed with 1 μl of antibody and 5 μl of 2×native RIPA buffer [0.5 M NaCl, 5% glycerol, 0.2 M Tris-HCl (pH 8.0),1.0% Tween-20, 2 mM EDTA] and incubated with rocking overnight at 4° C.For chimpanzee 1441 pre- and post-immune sera, precipitations wereperformed with the addition of recombinant protein G-coupled agarosebeads (Gibco), and incubation with rocking on ice for 1 h. For HEV#4,HEV#31 and HBV#8 Fab, a 1 μl of goat anti-human IgG (F(ab′)₂-specific)was used in addition to protein G-coupled agarose beads. The beads werepelleted, washed three times in 1× RIPA buffer and once with distilled,deionized H₂O (ddH₂O). Samples were then resuspended in 15 μL 2×Laemelli buffer and incubated for 10 min. at 95° C. prior to loading onto a 10-20% PAGE gel (Novex). After 1 h at 126 V, the gel was fixed in asolution of 10% acetone and 10% methanol for 20 min., washed twice indH₂O, then incubated in Amplify solution (Amersham) for 20 min. Afterdrying, the gel was exposed to X-ray film at −70° C.

RESULTS

[0096] Isolation and Characterization of HEV ORF 2-specific Fabs

[0097] Chimpanzee 1441 had been previously experimentally infected withthe HEV SAR-55 (Pakistan strain, Asian/African genotype). Prior to thebone marrow aspiration, the chimpanzee was immunized once withbaculovirus-expressed SAR-55 ORF 2 (55 kD) protein. Total RNA wasextracted from bone marrow lymphocytes. Messenger RNA was reversetranscribed using an oligo (dT) primer to generate cDNA. Amplificationof the cDNA was carried out by PCR using both κ-chain and γ1-chainprimers specific for the human antibody genes. The amplified κ- andγ1-chain genes were purified and cloned into the phage display vector,pComb3H. The resultant Fab phage library was then selected againstbaculovirus-expressed SAR-55 ORF 2 protein. After four rounds ofpanning, the library DNA was isolated and the phage display vectormodified by restriction enzyme digestion to allow for soluble Fabexpression in E. coli. An ELISA was used to determine the specificity ofthe Fabs using the HEV ORF 2 protein and a panel of unrelated proteinantigens. Of the 144 clones screened, seven were SAR-55 ORF2-specific.

[0098] As the restriction enzyme Bst N1 cuts frequently in the humanγ1-heavy chain [Marks, 1991], the resulting restriction patterns can beused to predict the presence of different heavy chain sequences amongstthe Fab clones. There were two distinct Bst N1 restriction patternsobserved, one represented by five clones of HEV#4, and the other by twoclones of HEV#31 (FIG. 1a).

[0099] Sequence analysis of the seven Fab clones confirmed the resultsof the Bst N1 digest above. There were two distinct γ1-heavy chains; onewas represented by HEV#4 clones and the other by HEV#31 clones. The twoγ1-chains varied markedly in all three complementarity-determiningregions (CDR; FIG. 1b). The κ-light chain sequences were also divergent(FIG. 7).

[0100] The specific germ-line origin of the two monoclonal antibodieswas assessed by conducting a sequence similarity search of all the knownhuman immunoglobulin genes. The two γ1-heavy chain sequences exhibitedthe most homology with the human VH3 family of germ line segments (Table1). HEV#4 was most closely related to DA-8 [Cook, 1994] VH gene segment,with 89.4% overall homology and 92% excluding CDR1 and CDR2. HEV#31 wasmost closely related to DP-47 [Tomlinson, 1992] VH gene segment, with88.5% overall homology and 91.7% excluding CDR1 and CDR2. The κ-lightchain sequences exhibited the most homology with the human Vκ1 family ofgerm line segments.

[0101] The affinities of the monoclonal antibodies were determined usingBIAcore™. Association and dissociation kinetics were measured for bothHEV#4 and HEV#31 binding to SAR-55 ORF2 protein (Table 2). Bothmonoclonal antibodies had high equilibrium dissociation constants(K_(d)), 1.7 nM for HEV#4 and 4.5 nM for HEV#31. TABLE 1 V_(H) V_(H) DJ_(H) V_(κ) V_(κ) J_(κ) Fab family segment segment segment familysegment segment HEV VH3 DA-8 ND* JH4b Vκ1 HK137 Jκ1 #4 HEV VH3 DP-47 ND*JH4b Vκ1 DPK9 Jκ4 #31

[0102] TABLE 2 MAb K_(d) k_(a) k_(d) HEV#4 1.7 1.2 3.5 HEV#31 4.5 0.544.9

[0103] A Western blot was performed to determine the nature of theepitopes recognized by the two monoclonal antibodies (i.e. linear orconformational epitopes). HEV#4 and HEV#31 both recognized reduced,denatured HEV ORF2 protein (FIG. 2), suggesting that they are bothdirected to linear epitopes on the virus capsid.

[0104] An indirect competition assay was performed to determine whetherthe two monoclonal antibodies recognized similar or overlapping epitopeson the HEV capsid (Table 3). Unlabeled HEV#4 blocked the binding ofbiotinylated HEV#31 to the SAR-55 ORF2 protein, and vice versa.Therefore, HEV#4 and HEV#31 recognized similar or overlapping epitopeson the SAR-55 ORF2 protein. TABLE 3 Biotinylated Fab Unlabeled Fab HEV#4HEV#31 HEV#4 63* 66 HEV#31 72  81

[0105] Radioimmunoprecipitation assays were carried out to determine thelocation of the epitopes on the HEV capsid. Purified HEV#4 and HEV#31were incubated at 4° C. overnight with a pool of six ³⁵S-labeledC-terminal truncated ORF2 translation products, shown schematically inFIG. 3a. Both monoclonal antibodies precipitated SAR-55 aa607 (FIG. 3b),corresponding to the 55 kD panning antigen. However, the shorterpolypeptides were not precipitated to any significant degree. Chimpanzee1441 immune serum precipitated SAR-55 aa308 to SAR-55 aa607, whilst thepre-immune serum did not react with any. SAR-55 aa208 was too poorlyradiolabeled to determine whether it was precipitated. HAV#6, anHAV-specific monoclonal antibody, did not precipitate any of the ORF2truncations, nor did the secondary antibody alone or protein G alone(FIG. 3b).

[0106] Currently, only one serotype of HEV is known. However, there area number of divergent strains of HEV based on nucleotide and amino acidsequences. Two of the most divergent strains of HEV are Pakistan(SAR-55) and swine HEV. The two Fabs were tested by ELISA forcross-reactivity with the swine HEV ORF2 protein. Titration curves forHEV#4 and HEV#31 were identical for the heterologous swine andhomologous SAR-55 ORF2 proteins (FIG. 4).

In Vitro Neutralization of HEV with in Vivo Monitoring in Rhesus Monkeys

[0107] Sixty four 50% monkey infectious doses (MID₅₀) of HEV strainSAR-55 were incubated with HEV#4, HEV#31, or an irrelevant Fab HBV#8, at1.9 mg ml⁻¹ , or with a 10% solution of either chimpanzee 5835 pre- orhyper-immune serum prior to inoculation into rhesus monkeys.Inoculations were performed in duplicate. After intravenous inoculation,the monkeys were followed for 20 weeks for biochemical evidence ofhepatitis (serum ALT) and for seroconversion to HEV antigens by ELISA.All the monkeys that received HEV incubated either with chimpanzee 5835pre-immune serum or HBV#8 were infected and developed hepatitis, asevidenced by a rise in ALT levels and seroconversion to HEV ORF2 protein(examples shown in FIG. 5a, b). In contrast, all monkeys receiving HEVincubated with HEV#4, HEV#31 or chimpanzee 5835 hyper-immune serum hadnormal ALT levels and did not seroconvert to HEV ORF2 protein (examplesshown in FIG. 5c-e).

[0108] Antibodies EBL#2 and EBL#89 (listed in FIG. 8) were tested fortheir ability to neutralize HEV (SAR-55 strain) by mixing virus andantibody in vitro as described above and monitoring for residualinfectivity by intravenous inoculation of rhesus monkeys. AntibodiesEBL#2 and EBL#89 were selected because they did not inhibit binding ofeach other to HEV ORF2 protein. Nor did they inhibit binding of the twoneutralizing MAbs (HEV#4 and HEV#31) to the HEV ORF2 protein (FIG. 9).Therefore, each was directed to a unique non-overlapping epitope on theHEV ORF2 protein. Panel (A) shows the serum ALT profile over 15 weeks offollow-up for one of the two animals receiving HEV (SAR-55 strain) mixedwith EBL#2. The rise in ALT at week 6 and subsequent seroconversion toanti-HEV (IgM and IgG) indicated that this antibody did not neutralizeHEV (i.e. the epitope it recognizes is a non-neutralization epitope).Panel (B) shows the serum ALT profile over 15 weeks of follow-up for oneof the two animals receiving HEV (SAR-55 strain) mixed with EBL#89. Therise in ALT at week 10 and seroconversion to anti-HEV (IgM and IgG)indicated that this antibody did not neutralize HEV (i.e. the epitope itrecognizes is a non-neutralization epitope). Panel (C) is a positivecontrol hyper-immune serum which does neutralize the SAR-55 inoculum.

[0109] The MAbs listed in FIG. 8 were used in pair-wise competitionELISAs to determine the topography of the epitopes recognized by theseantibodies. The data is summarized in FIG. 10. Where two circles overlapthere is >50% inhibition of binding between the antibody pair. However,the degree of overlap does not represent the percentage inhibition ofbinding between each antibody pair. The data indicate that all the MAbsare directed to a single antigenic site on the HEV ORF2 protein. Thatantigenic site comprises overlapping and non-overlapping epitopes.

[0110] Both C-terminally and N-terminally truncated forms of the SAR-55ORF2 55 kD protein (amino acids 112-607) were constructed in order tomap the location of the epitopes on the ORF2 protein recognized by thepanel of MAbs listed in Table 4 and FIG. 8. FIG. 11 shows the data fromradioimmunoprecipitation studies overlaid on top of the topographicalmap of the HEV ORF2 antigenic site. Analysis of the data indicated thatfour MAbs recognized epitopes located at the N-terminal portion of theHEV ORF2 55 kD protein (between amino acids 112 and 208). Six MAbsmapped to the C-terminal portion of the 55 kD protein (between aminoacids 578 and 607). This region contains the neutralization epitopes.Four MAbs are unresolved by this study. Three MAbs, which could not bemapped by pair-wise competition assays, also recognized epitopes in theC-terminal portion of the HEV ORF2 55 kD protein. These results aresummarized in Table 4. FIG. 11 depicts the location of theneutralization and non-neutralization epitopes on the ORF2 protein.TABLE 4 Mab Epitope Location Activity HEV#4 578-607 Neutralizing HEV#31578-607 Neutralizing ERL#1 578-607 EBL#2 unresolved Non-neutralizingEBL#3 unresolved EBL#4 578-607 EBL#5 unresolved EBL#8 112-208 EBL#9578-607 EBL#10 unresolved EBL#16 578-607 EBL#33 578-607 EBL#53 578-607EBL#56 578-607 EBL#77 112-208 EBL#79 112-208 EBL#89 112-208Non-neutralizing

[0111] There are at least three genotypes of HEV: genotype 1 comprisingstrains from Asia and Africa, genotype 2 comprising the Mexican strain,and genotype 3 comprising the human US strains and the swine HEV, Mengstrain. To determine whether this panel of MAbs could recognize ORF2proteins from other genotypes, we initially performed a qualitativeELISA. All of the MAbs tested (16 of 17) recognized the ORF2 proteinfrom the homologous strain SAR-55, and also recognized the ORF2 proteinfrom the genotype 3 swine HEV Meng strain (data not shown). Subsequentlya quantitative measurement of antibody binding was undertaken. Theaffinities of these MAbs for the SAR-55 and Meng ORF2 proteins weredetermined. For fifteen of sixteen MAbs, the affinity values werecomparable indicating conservation of epitopes between the two strains.However, one MAb, EBL#16 had a >1000-fold reduction in affinity for theMeng strain ORF2 protein compared to that for the SAR-55 ORF2 protein,thus indicating that an amino acid(s) substitution in or near thisepitope is responsible for the reduction in affinity. Since EBL#16precipitated C-607 only, it is likely that the epitope recognized liesbetween about aa578 and about aa607 of the ORF2 protein. A comparison ofthe amino acid sequences in this region for a number of HEV isolates isshown in FIG. 6 with SAR-55 and Meng strains highlighted. There are only5 amino acid differences between the two strains. Hence it is likelythat one or more of these amino acid changes is responsible for thereduction in affinity for the Meng ORF2 protein. Thus, it is possible tomake diagnostic assays that distinguish between infection with swine HEVand some human HEV strains.

[0112] It is also interesting to note that the epitopes recognized bythe two neutralizing MAbs HEV#4 and HEV#31 are conserved between the twodivergent HEV strains. If these neutralization sites are conserved inother strains of HEV then these antibodies would be broadly effective inpassive immunoprophylaxis and immunotherapy.

[0113] The affinities of all of the MAbs for both SAR-55 ORF2 protein(homologous strain) and the swine HEV, Meng strain (heterologous strain)were determined by competition inhibition ELISA. The concentration offree ORF2 protein required to inhibit antibody binding by 50% isequivalent to the equilibrium dissociation constant (K_(d)). The K_(d)values for the Fab are summarized in Table 5. TABLE 5 Kd (nM) SAR-55Swine MAb ORF2 ORF2 HEV#4 3.3 7.0 HEV#31 0.8 1.3 EBL#1 15.0 22.5 EBL#22.0 4.0 EBL#3 3.0 4.8 EBL#4 ND^(#) ND^(#) EBL#5 1.1 3.0 EBL#8 0.7 1.8EBL#9 45.0 35.0 EBL#10 2.0 3.0 EBL#16 1.3 >1000* EBL#33 >1000* >1000*ERL#53 400.0 ≧1000* EBL#56 48.0 45.0 EBL#77 1.9 4.0 EBL#79 4.0 8.5EBL#89 2.0 3.3

DISCUSSION

[0114] Antibodies to a wide range of viral pathogens have been isolatedusing combinatorial antibody libraries displayed on the surface offilamentous phage particles. In most studies, human donors infected withspecific viral pathogens have been used as the source of bone marrowcells or peripheral blood lymphocytes for the construction of theselibraries. In some studies, “naïve” libraries have been constructedusing uninfected donors [Marks, 1991]. In the present invention, achimpanzee previously infected with specific viral pathogens was used asa source of bone marrow lymphocytes for the construction of a phagedisplay library. The advantages of using a chimpanzee as a donor forrepertoire cloning are two-fold: first, the chimpanzee can be infectedby many of the important human viral pathogens with limited host range,e.g. HIV-1, HCV, HBV, and RSV; second, as the chimpanzee is the primatemost closely related to humans, chimpanzee antibodies couldtheoretically be used directly in the immune prophylactic treatment ofhuman diseases. A number of studies have addressed the possibility ofusing primate reagents in human prophylaxis and therapy by examining thereverse situation, i.e. introduction of human immune components intoprimates [Logdberg, 1994; Ehrlich, 1988; Ehrlich, 1988; Ehrlich, 1987;Ehrlich, 1990]. The data from those studies show that littleimmunogenicity is seen when human immune components are introduced intochimpanzees compared to other primates.

[0115] The cDNA phage display library described herein is a potentialrepertoire for antibodies to the five recognized hepatitis-causingviruses, HAV, HBV, HCV, HDV and HEV. In the initial study, twoHEV-specific monoclonal antibodies directed to the ORF2 protein wereidentified. The γ1-heavy chains of those two monoclonal antibodies sharea high degree of homology (89.4% for HEV#4 and 88.5% for HEV#31) at thenucleotide level with two different γ1-heavy chains from the human VH3gene family. The degree of homology between the chimpanzee and humanγ-chain genes was similar to that of the only other chimpanzee antibodycharacterized to date. For an anti-HIV gp160 monoclonal antibody, therewas 92% homology with its nearest human germ line equivalent. This wasestimated to be more homologous than the two most distantly relatedhuman VH gene families [Vijh-Warrier, 1995]. Such close sequencehomology between chimpanzee and human antibody genes suggests thatchimpanzee antibodies could be useful in human immunotherapy withoutmodification (“humanization”). However, as with human monoclonalantibodies, each monoclonal antibody would require testing forimmunogenicity in humans.

[0116] HEV#4 and HEV#31 have high affinities for the ORF2 protein fromHEV strain SAR-55, with K_(d) values in the nanomolar range. Thesevalues were comparable to K_(d) values determined for other neutralizingFabs to other viruses, e.g. influenza A virus [Schofield, 1996], HIV-1[Burton, 1994], and murine hepatitis virus [Lamarre, 1995]. In Westernblot, both HEV#4 and HEV#31 recognized reduced, denatured ORF2suggesting that they are directed to linear rather than conformationalepitopes on the ORF2 protein. In indirect competition assays, HEV#4 andHEV#31 recognized similar or overlapping epitopes on the ORF2 proteinsince each Fab inhibited the other from binding. The location of thisepitope or epitopes was determined by radioimmunoprecipitation ofC-terminally truncated SAR-55 ORF2 proteins. HEV#4 and HEV#31 stronglyprecipitated only the construct corresponding to aa112-607 (55 kDprotein), suggesting that the majority of the epitope(s) lies betweenaa578 and aa607 on the ORF2 protein. This epitope(s) forms part of theantigenic region 6 designated by Khudyakov et al. [Khudyakov, 1999]. Theweakly precipitated ORF2 truncation products aa112 to aa308, aa112 toaa408, aa112 to aa508 and aa112 to aa578 probably represent non-specificprotein-protein interactions. The amino acid sequence between aa578 andaa607 is relatively conserved amongst HEV isolates (FIG. 6), with theMexican strain having the most amino acid changes, 5 out of 30.Reactivity of the two monoclonal antibodies with recombinant ORF2protein from a highly divergent heterologous strain, swine HEV, wasdetermined by ELISA. Both monoclonal antibodies had similar titrationcurves with the SAR-55 ORF2 and swine ORF2 proteins. Since this regionis relatively well conserved, it is conceivable that the epitope(s)recognized by HEV#4 and HEV#31 are likely to be conserved amongst manyof the different HEV isolates. Currently, cloning of the ORF2 from theMexico strain is being attempted in order to determine if the epitope(s)is conserved in this region of ORF2 from the most divergent strain.

[0117] Neutralization of the SAR-55 strain of HEV by monoclonalantibodies HEV#4 and HEV#31 was determined by intravenous challenge ofrhesus monkeys with 64 MID₅₀ after incubation of the virus with the twomonoclonal antibodies. All the animals receiving HEV incubated witheither HEV#4 or HEV#31 did not seroconvert to anti-HEV, nor was any risein serum ALT levels detected. In contrast, all control animals wereinfected with HEV since they seroconverted to anti-HEV, and also hadmild ALT elevations. Therefore, both HEV#4 and HEV#31 neutralized HEV.Furthermore, since the Fabs are monovalent, neutralization of HEV wasnot due to a reduction in the infectious dose given to the monkeys dueto the aggregation of virus particles. Neutralization of HEV was causedby the binding of the monoclonal antibodies alone, since the Fabfragments which lack an Fc region would not be able to neutralize thevirus by an Fc-mediated function, such as antibody-dependent cellmediated cytotoxicity.

[0118] Currently, here is no vaccine available for the prevention of HEVinfection. Therefore, there is a need for anti-HEV immunoglobulins whichcan be used for protecting individuals at high risk from HEV infection.Since currently such therapies are very expensive, economically viableand renewable sources of potent IgGs would be very beneficial. At thepresent, the production of antibodies generated from stably transfectedcell lines is still prohibitively expensive. However, new techniquessuch as the expression of whole IgG molecules in plants [Ma, 1998] maymake these antibodies cheaper to produce, and economically viable. Inaddition to being a potential source of antibodies for passiveimmunoprophylaxis, this cDNA library described herein could also providea repository of antibodies which may be helpful in elucidating the typeof antibodies successful vaccines should be stimulating.

References

[0119] 1. Arankalle, V. A., Goverdhan, M. K., Banerjee, K. 1994.Antibodies against hepatitis E virus in old world monkeys. Journal ofViral Hepatitis. 1:125-129.

[0120] 2. Aye, T. T., T. Uchida, X. Z. Ma, F. Iida, T. Shikata, H.Zhuang, and K. M. Win. 1992. Complete nucleotide sequence of a hepatitisE virus isolated from the Xinjiang epidemic (1986-1988) of China.Nucleic Acids Res. 20:3512.

[0121] 3. Barbas, C. F. d., A. S. Kang, R. A. Lerner, and S. J.Benkovic. 1991. Assembly of combinatorial antibody libraries on phagesurfaces: the gene III site. Proc Natl Acad Sci U S A. 88:7978-82.

[0122] 4. Bender, E., G. R. Pilkington, and D. R. Burton. 1994. Humanmonoclonal Fab fragments from a combinatorial library prepared from anindividual with a low serum titer to a virus. Hum Antibodies Hybridomas.5:3-8.

[0123] 5. Bender, E., J. M. Woof, J. D. Atkin, M. D. Barker, C. RBebbington, and D. R. Burton. 1993. Recombinant human antibodies:linkage of an Fab fragment from a combinatorial library to an Fcfragment for expression in mammalian cell culture. Hum AntibodiesHybridomas. 4:74-9.

[0124] 6. Bi, S. L., M. A. Purdy, K. A. McCaustland, H. S. Margolis, andD. W. Bradley. 1993. The sequence of hepatitis E virus isolated directlyfrom a single source during an outbreak in China [published erratumappears in Virus Res July 1994;33(1):98]. Virus Res. 28:233-47.

[0125] 7. Burton, D. R., and C. F. Barbas, 3rd. 1994. Human antibodiesfrom combinatorial libraries. Adv Immunol. 57:191-280.

[0126] 8. Burton, D. R., C. F. d. Barbas, M. A. Persson, S. Koenig, R.M. Chanock, and R. A. Lerner. 1991. A large array of human monoclonalantibodies to type 1 human immunodeficiency virus from combinatoriallibraries of asymptomatic seropositive individuals. Proc Natl Acad Sci US A. 88:10134-7.

[0127] 9. Burton, D. R., J. Pyati, R. Koduri, S. J. Sharp, G. B.Thornton, P. W. Parren, L. S. Sawyer, R. M. Hendry, N. Dunlop, P. L.Nara, and et al. 1994. Efficient neutralization of primary isolates ofHIV-1 by a recombinant human monoclonal antibody. Science. 266:1024-7.

[0128] 10. Clayson, E. T., B. L. Innis, K. S. Myint, S. Narupiti, D. W.Vaughn, S. Giri, P. Ranabhat, and M. P. Shrestha. 1995. Detection ofhepatitis E virus infections among domestic swine in the KathmanduValley of Nepal. Am J Trop Med Hyg. 53:228-32.

[0129] 11. Cook, G. P., and I. M. Tomlinson. 1995. The humanimmunoglobulin VH repertoire. Immunol Today. 16:237-42.

[0130] 12. Cook, G. P., I. M. Tomlinson, G. Walter, H. Riethman, N. P.Carter, L. Buluwela, G. Winter, and T. H. Rabbitts. 1994. A map of thehuman immunoglobulin VH locus completed by analysis of the telomericregion of chromosome 14q. Nat Genet. 7:162-8.

[0131] 13. Crowe, J. E., Jr., B. R. Murphy, R. M. Chanock, R. A.Williamson, C. F. Barbas, 3rd, and D. R. Burton. 1994. Recombinant humanrespiratory syncytial virus (RSV) monoclonal antibody Fab is effectivetherapeutically when introduced directly into the lungs of RSV-infectedmice. Proc Natl Acad Sci U S A. 91:1386-90.

[0132] 14. de Kruif, J., A. R. van der Vuurst de Vries, L. Cilenti, E.Boel, W. van Ewijk, and T. Logtenberg. 1996. New perspectives onrecombinant human antibodies. Immunol Today. 17:453-5.

[0133] 15. Ditzel, H. J., P. W. Parren, J. M. Binley, J. Sodroski, J. P.Moore, C. F. Barbas, 3rd, and D. R. Burton. 1997. Mapping the proteinsurface of human immunodeficiency virus type 1 gp120 using humanmonoclonal antibodies from phage display libraries. J Mol Biol.267:684-95.

[0134] 16. Donati, M. C., Fagan, E. A., and Harrison, T. J. 1997.Sequence analysis of full length HEV clones derived directly from humanliver in fulminant hepatitis E., p. 313-316. In M. Rizzetto, Purcell, R.H., Gerin, J. L., and Verme, G. (ed.), Viral Hepatitis and LiverDisease. Edizioni Minervva Medica, Torino.

[0135] 17. Ehrlich, P. H., K. E. Harfeldt, J. C. Justice, Z. A.Moustafa, and L. Ostberg. 1987. Rhesus monkey responses to multipleinjections of human monoclonal antibodies. Hybridoma. 6:151-60.

[0136] 18. Ehrlich, P. H., Z. A. Moustafa, K. E. Harfeldt, C. Isaacson,and L. Ostberg. 1990. Potential of primate monoclonal antibodies tosubstitute for human antibodies: nucleotide sequence of chimpanzee Fabfragments. Hum Antibodies Hybridomas. 1:23-6.

[0137] 19. Ehrlich, P. H., Z. A. Moustafa, J. C. Justice, K. E.Harfeldt, I. K. Gadi, L. J. Sciorra, F. P. Uhl, C. Isaacson, and L.Ostberg. 1988. Human and primate monoclonal antibodies for in vivotherapy. Clin Chem. 34:1681-8.

[0138] 20. Ehrlich, P. H., Z. A. Moustafa, J. C. Justice, K. E.Harfeldt, and L. Ostberg. 1988. Further characterization of the fate ofhuman monoclonal antibodies in rhesus monkeys. Hybridoma. 7:385-95.

[0139] 21. Geoffroy, F., R. Sodoyer, and L. Aujame. 1994. A new phagedisplay system to construct multicombinatorial libraries of very largeantibody repertoires. Gene. 151:109-13.

[0140] 22. Glamann, J., D. R. Burton, P. W. Parren, H. J. Ditzel, K. A.Kent, C. Arnold, D. Montefiori, and V. M. Hirsch. 1998. Simianimmunodeficiency virus (SIV) envelope-specific Fabs with high-levelhomologous neutralizing activity: recovery from along-term-nonprogressor SIV-infected macaque. J Virol. 72:585-92.

[0141] 23. Huang, C. C., D. Nguyen, J. Fernandez, K. Y. Yun, K. E. Fry,D. W. Bradley, A. W. Tam, and G. R. Reyes. 1992. Molecular cloning andsequencing of the Mexico isolate of hepatitis E virus (HEV). Virology.191:550-8.

[0142] 24. Joshi, Y. K., S. Babu, S. Sarin, B. N. Tandon, B. M. Gandhi,and V. C. Chaturvedi. 1985. Immunoprophylaxis of epidemic non-A non-Bhepatitis. Indian J Med Res. 81:18-9.

[0143] 25. Kabrane-Lazizi, Y., X. J. Meng, R. H. Purcell, and S. U.Emerson. 1999. Evidence that the genomic RNA of hepatitis E virus iscapped. J Virol. 73:8848-50.

[0144] 26. Karetnyi, V., D. I. Dzhumalieva, R. K. Usmanov, I. P. Titova,I. Litvak Ia, and M. S. Balaian. 1993. [The possible involvement ofrodents in the spread of viral hepatitis E]. Zh Mikrobiol EpidemiolImmunobiol:52-6.

[0145] 27. Khudyakov, Y. E., E. N. Lopareva, D. L. Jue, T. K. Crews, S.P. Thyagarajan, and H. A. Fields. 1999. Antigenic domains of the openreading frame 2-encoded protein of hepatitis E virus. J Clin Microbiol.37:2863-71.

[0146] 28. Khuroo, M. S., and M. Y. Dar. 1992. Hepatitis E: evidence forperson-to-person transmission and inability of low dose immune serumglobulin from an Indian source to prevent it [see comments]. Indian JGastroenterol. 11:113-6.

[0147] 29. Khuroo, M. S., M. R. Teli, S. Skidmore, M. A. Sofi, and M. I.Khuroo. 1981. Incidence and severity of viral hepatitis in pregnancy. AmJ Med. 70:252-5.

[0148] 30. Laemelli, E. K. 1970. Cleavage of structural proteins duringthe assembly of the head of bacteriophage T4. Nature. 227:680-685.

[0149] 31. Lamarre, A., and P. J. Talbot. 1995. Protection from lethalcoronavirus infection by immunoglobulin fragments. J Immunol.154:3975-84.

[0150] 32. Logdberg, L., E. Kaplan, M. Drelich, E. Harfeldt, H. Gunn, P.Ehrlich, D. Dottavio, P. Lake, and L. Ostberg. 1994. Primate antibodiesto components of the human immune system. J Med Primatol. 23:285-97.

[0151] 33. Ma, J. K., B. Y. Hikmat, K. Wycoff, N. D. Vine, D.Chargelegue, L. Yu, M. B. Hein, and T. Lehner. 1998. Characterization ofa recombinant plant monoclonal secretory antibody and preventiveimmunotherapy in humans [see comments]. Nat Med. 4:601-6.

[0152] 34. Marks, J. D., H. R. Hoogenboom, T. P. Bonnert, J. McCafferty,A. D. Griffiths, and G. Winter. 1991. By-passing immunization. Humanantibodies from V-gene libraries displayed on phage. J Mol Biol.222:581-97.

[0153] 35. Mast, E. E., Alter, M. J. 1993. Epidemiology of viralhepatitis: an overview. Seminars in Virology. 4:273-283.

[0154] 36. Mast, E. E., Kuramoto, P. O., Favorov M. O., Schoening, V.R., Burkholder, B. T., Shapiro, C. N., Holland, P. V. 1997. Prevelanceof and risk factors for antibody to hepatitis E virus seroreactivityamong blood donors in northern California. Journal of InfectiousDiseases. 176:34-40.

[0155] 37. Meng, X. J., Purcell, R. H., Halbur, P. G., Lehman, J. R.,Webb, D. M., Tsareva, T. S., Haynes, J. S., Thacker, B. J., Emerson, S.U. 1997. A novel virus in swine is closely related to the humanhepatitis E virus. PNAS. 94:9860-9865.

[0156] 38. Ogata, N., L. Ostberg, P. H. Ehrlich, D. C. Wong, R. H.Miller, and R. H. Purcell. 1993. Markedly prolonged incubation period ofhepatitis B in a chimpanzee passively immunized with a human monoclonalantibody to the a determinant of hepatitis B surface antigen. Proc NatlAcad Sci U S A. 90:3014-8.

[0157] 39. Panda, S. K., S. K. Nanda, M. Zafrullah, I. H. Ansari, M. H.Ozdener, and S. Jameel. 1995. An Indian strain of hepatitis E virus(HEV): cloning, sequence, and expression of structural region andantibody responses in sera from individuals from an area of high-levelHEV endemicity. J Clin Microbiol. 33:2653-9.

[0158] 40. Persson, M. A., R. H. Caothien, and D. R. Burton. 1991.Generation of diverse high-affinity human monoclonal antibodies byrepertoire cloning. Proc Natl Acad Sci U S A. 88:2432-6.

[0159] 41. Purcell, R. H. 1996. Hepatitis E Virus. In B. N. Fields,Knipe, D. M., & Howley, P. M. (ed.), Fields Virology, 3rd ed.Lippinscott-Raven, Philadelphia.

[0160] 42. Robinson, R. A., W. H. Burgess, S. U. Emerson, R. S.Leibowitz, S. A. Sosnovtseva, S. Tsarev, and R. H. Purcell. 1998.Structural characterization of recombinant hepatitis E virus ORF2proteins in baculovirus-infected insect cells. Protein Expr Purif.12:75-84.

[0161] 43. Schlauder, G. G., G. J. Dawson, J. C. Erker, P. Y. Kwo, M. F.Knigge, D. L. Smalley, J. E. Rosenblatt, S. M. Desai, and I. K.Mushahwar. 1998. The sequence and phylogenetic analysis of a novelhepatitis E virus isolated from a patient with acute hepatitis reportedin the United States [published erratum appears in J Gen Virol Octber1998;79(Pt 10):2563]. J Gen Virol. 79:447-56.

[0162] 44. Schofield, D. J., and N. J. Dimmock. 1996. Determination ofaffinities of a panel of IgGs and Fabs for whole enveloped (influenza A)virions using surface plasmon resonance. J Virol Methods. 62:33-42.

[0163] 45. Tam, A. W., M. M. Smith, M. E. Guerra, C. C. Huang, D. W.Bradley, K. E. Fry, and G. R. Reyes. 1991. Hepatitis E virus (HEV):molecular cloning and sequencing of the full-length viral genome.Virology. 185:120-31.

[0164] 46. Thomas, D. L., P. O. Yarbough, D. Vlahov, S. A. Tsarev, K. E.Nelson, A. J. Saah, and R. H. Purcell. 1997. Seroreactivity to hepatitisE virus in areas where the disease is not endemic. J Clin Microbiol.35:1244-7.

[0165] 47. Thompson, J., T. Pope, J. S. Tung, C. Chan, G. Hollis, G.Mark, and K. S. Johnson. 1996. Affinity maturation of a high-affinityhuman monoclonal antibody against the third hypervariable loop of humanimmunodeficiency virus: use of phage display to improve affinity andbroaden strain reactivity. J Mol Biol. 256:77-88.

[0166] 48. Tomlinson, I. M., G. Walter, J. D. Marks, M. B. Llewelyn, andG. Winter. 1992. The repertoire of human germline VH sequences revealsabout fifty groups of VH segments with different hypervariable loops. JMol Biol. 227:776-98.

[0167] 49. Tsarev, S. A., S. U. Emerson, G. R. Reyes, T. S. Tsareva, L.J. Legters, I. A. Malik, M. Iqbal, and R. H. Purcell. 1992.Characterization of a prototype strain of hepatitis E virus. Proc NatlAcad Sci U S A. 89:559-63.

[0168] 50. Tsarev, S. A., T. S. Tsareva, S. U. Emerson, S. Govindarajan,M. Shapiro, J. L. Gerin, and R. H. Purcell. 1997. Recombinant vaccineagainst hepatitis E: dose response and protection against heterologouschallenge. Vaccine. 15:1834-8.

[0169] 51. Tsarev, S. A., T. S. Tsareva, S. U. Emerson, S. Govindarajan,M. Shapiro, J. L. Gerin, and R. H. Purcell. 1994. Successful passive andactive immunization of cynomolgus monkeys against hepatitis E. Proc NatlAcad Sci U S A. 91:10198-202.

[0170] 52. Tsarev, S. A., T. S. Tsareva, S. U. Emerson, A. Z. Kapikian,J. Ticehurst, W. London, and R. H. Purcell. 1993. ELISA for antibody tohepatitis E virus (HEV) based on complete open-reading frame-2 proteinexpressed in insect cells: identification of HEV infection in primates.J Infect Dis. 168:369-78.

[0171] 53. Tsarev, S. A., T. S. Tsareva, S. U. Emerson, P. O. Yarbough,L. J. Legters, T. Moskal, and R. H. Purcell. 1994. Infectivity titrationof a prototype strain of hepatitis E virus in cynomolgus monkeys. J MedVirol. 43:135-42.

[0172] 54. Tsega, E., B. G. Hansson, K. Krawczynski, and E. Nordenfelt.1992. Acute sporadic viral hepatitis in Ethiopia: causes, risk factors,and effects on pregnancy. Clin Infect Dis. 14:961-5.

[0173] 55. Vijh-Warrier, S., E. Murphy, I. Yokoyama, and S. A. Tilley.1995. Characterization of the variable regions of a chimpanzeemonoclonal antibody with potent neutralizing activity against HIV-1. MolImmunol. 32:1081-92.

[0174] 56. Williamson, R. A., R. Burioni, P. P. Sanna, L. J. Partridge,C. F. d. Barbas, and D. R. Burton. 1993. Human monoclonal antibodiesagainst a plethora of viral pathogens from single combinatoriallibraries [published erratum appears in Proc Natl Acad Sci U S A Feb. 1,1994;91(3):1193]. Proc Natl Acad Sci U S A. 90:4141-5.

[0175] 57. Winter, G., A. D. Griffiths, R. E. Hawkins, and H. R.Hoogenboom. 1994. Making antibodies by phage display technology. AnnuRev Immunol. 12:433-55.

[0176] 58. Yin, S., R. H. Purcell, and S. U. Emerson. 1994. A newChinese isolate of hepatitis E virus: comparison with strains recoveredfrom different geographical regions. Virus Genes. 9:23-32.

[0177] 59. Zhuang, H., X. Y. Cao, C. B. Liu, and G. M. Wang. 1991.Epidemiology of hepatitis E in China. Gastroenterol Jpn. 26 Suppl3:135-8.

1 44 1 126 PRT Chimpanzee Antibody 1 Glu Val Gln Leu Leu Glu Ser Gly GlySer Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala AlaSer Gly Phe Thr Phe Ser Asp Ser 20 25 30 Trp Met His Trp Val Arg Gln ValPro Gly Lys Gly Leu Glu Trp Asx 35 40 45 Asp Thr Ile Ser Ser Asp Gly AspSer Thr Arg Tyr Ala Asp Ser Val 50 55 60 Gln Gly Arg Phe Ile Ile Ser ArgAsp Asn Ala Lys Asn Thr Tyr Leu 65 70 75 80 Leu Gln Met Asn Ser Leu ArgVal Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Pro Gln Tyr CysSer Thr Thr Arg Cys Asp Trp Ile His 100 105 110 Tyr Asp Tyr Trp Gly GlnGly Thr Leu Val Thr Val Ser Ser 115 120 125 2 112 PRT ChimpanzeeAntibody 2 Ala Glu Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp 1 5 10 15 Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Asp Val Gly HisTyr Leu 20 25 30 Gly Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Lys Arg LeuIle Tyr 35 40 45 Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe SerGly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu GlnPro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser TyrPro Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr ValAla Ala Pro 100 105 110 3 131 PRT Chimpanzee Antibody 3 Glu Val Gln LeuLeu Glu Ser Gly Gly Ser Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu ArgLeu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Asn His 20 25 30 Ala Ile HisTrp Val Arg Gln Thr Ser Asp Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr IleSer Gly Gly Gly Gly Ala Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly ArgGlu Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Val Tyr 65 70 75 80 Leu GlnMet Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala LysAsp Val Leu Asn Val Phe Glu Ala Glu Arg Asn Tyr Gly Trp 100 105 110 SerThr Gly Tyr Ser Phe Asp Tyr Trp Gly Gln Gly Thr Arg Val Thr 115 120 125Val Ser Ser 130 4 117 PRT Chimpanzee Antibody 4 Ala Glu Leu Gln Met ThrGln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val ThrIle Thr Cys Arg Ala Ser His Lys Met Tyr Asp 20 25 30 Tyr Val Ser Trp TyrHis Gln Arg Pro Gly Glu Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Ala Ala SerThr Leu Gln Thr Gly Ala Pro Thr Arg Phe Ser 50 55 60 Gly Ser Gly Ser GlyThr Asp Phe Thr Leu Thr Ile Gly Gly Leu Gln 65 70 75 80 Pro Glu Asp PheGly Thr Tyr Tyr Cys Gln Arg Ala Phe Gly Thr Gln 85 90 95 Leu Thr Phe GlyGly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro SerSer Ser 115 5 378 DNA Chimpanzee Antibody 5 gaggtgcagc tgctcgagtctgggggaagc ttagttcagc cgggggggtc cctgagactc 60 tcctgtgcag cctctggattcaccttcagt gattcgtgga tgcactgggt ccgccaagtc 120 ccagggaagg ggctggagtgggtctcacgt atcagtagtg atggcgacag cacaagatac 180 gcggactccg tgcagggccgattcatcatc tccagagaca acgccaagaa cacactgtat 240 ctgcagatga atagtctgagagtcgaggac acggctgtgt attattgcac aagatcgccg 300 caatattgta gtactaccaggtgcgactgg attcactatg actactgggg ccaggggacc 360 ctggtcaccg tctcctca 3786 338 DNA Chimpanzee Antibody 6 gccgagctca cccagtctcc atcctcactgtctgcatctg taggagacag agtcaccatc 60 acttgtcggg cgagtcagga cgttggccattatttaggct ggtttcagca gaaacctggg 120 caagccccta agcgcctgat ctatgctgcatccaatttgc agagtggggt cccatccagg 180 ttcagcggca gtggatctgg gacagaattcactctcacaa tcaggagcct ttcagcggca 240 gattttgcca cttattactg tctacaacataatagttacc cttggacgtt gattttgcca 300 accaagctgg aaatcaaacg aactgtggctgcaccatc 338 7 393 DNA Chimpanzee Antibody 7 gaggtgcagc tgctcgagtctgggggaggc ttggtacagc cgggggggtc cctaagactc 60 tcgtgtgcag cctctggattcatcttcagc aaccatgcca tacactgggt ccgccagact 120 tcagacaagg ggctggagtgggtcgcaact attagtggtg gtggtggtgc cacttattat 180 ccagactctg tcaagggccgattcaccatc tccagagaca attcgaagaa tatggtgtat 240 ctgcagatga acagcctgagagccgaggac acggccgtgt attactgtgc gaaagatgtt 300 ttaaatgttt tcgaggcggaacgaaactat ggttggagta ccgggtactc ctttgactac 360 tggggccagg gaacccgggtcaccgtctcc tca 393 8 351 DNA Chimpanzee Antibody 8 gccgagctcc agatgacccagtctccatcc tccctgtctg catctgtggg agacagagtc 60 actattacat gccgggcgagtcacaaaatg tacgactatg tgagttggta tcaccagaga 120 ccgggggaag cccctaggctcctgatctat gccgcctcaa ccttgcaaac tggggcccca 180 acaaggttca gtggcagtggatctgggaca gacttcactc tcaccatcgg cggtctgcaa 240 cctgaagatt ttggaacatattactgtcag cgtgctttcg ggacacagct caccttcggt 300 ggagggacca aggtggagatcaaacgaact gtggctgcac catcttcttc a 351 9 30 PRT Hepatitis E virus 9 ArgVal Ala Ile Ser Thr Tyr Thr Thr Arg Leu Gly Ala Gly Pro Val 1 5 10 15Ala Ile Ser Ala Ala Ala Val Leu Ala Pro Arg Ser Ala Leu 20 25 30 10 30PRT Hepatitis E virus 10 Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu GlyAla Gly Pro Val 1 5 10 15 Ser Ile Ser Ala Val Ala Val Leu Ala Pro HisSer Ala Leu 20 25 30 11 30 PRT Hepatitis E virus 11 Arg Val Ala Ile SerThr Tyr Thr Thr Ser Leu Gly Ala Gly Pro Val 1 5 10 15 Ser Ile Ser AlaVal Ala Val Leu Ala Pro His Ser Val Leu 20 25 30 12 30 PRT Hepatitis Evirus 12 Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro Val1 5 10 15 Ser Ile Ser Ala Val Ala Val Leu Thr Pro His Ser Ala Leu 20 2530 13 30 PRT Hepatitis E virus 13 Arg Val Ala Ile Ser Thr Tyr Thr ThrSer Leu Gly Ala Gly Pro Val 1 5 10 15 Ala Ile Ser Ala Val Ala Val LeuAla Pro His Ser Ala Leu 20 25 30 14 30 PRT Hepatitis E virus 14 Arg ValAla Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro Val 1 5 10 15 SerIle Ser Ala Val Ala Val Leu Gly Pro His Ser Ala Leu 20 25 30 15 30 PRTHepatitis E virus 15 Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly AlaGly Pro Thr 1 5 10 15 Ser Ile Ser Ala Val Gly Val Leu Ala Pro His SerAla Leu 20 25 30 16 17 PRT Chimpanzee Antibody 16 Ser Pro Gln Tyr CysSer Thr Thr Arg Cys Asp Trp Ile His Tyr Asp 1 5 10 15 Tyr 17 22 PRTChimpanzee Antibody 17 Asp Val Leu Asn Val Phe Glu Ala Glu Arg Asn TyrGly Trp Ser Thr 1 5 10 15 Gly Tyr Ser Phe Asp Tyr 20 18 6 PRT ChimpanzeeAntibody 18 Gly Asn Ser Leu Asp Tyr 1 5 19 18 PRT Chimpanzee Antibody 19Gly Met Glu Tyr Tyr Asp Asn Trp Gly Lys Val Phe Leu Asp Ala Phe 1 5 1015 Asp Leu 20 18 PRT Chimpanzee Antibody 20 Ser Glu Val Gly Gly Ser TrpTyr Ile Asp Val Glu Ser Asn Trp Phe 1 5 10 15 Asp Pro 21 8 PRTChimpanzee Antibody 21 Glu His Trp Arg Gln Leu Asp Tyr 1 5 22 18 PRTChimpanzee Antibody 22 Gly Asp Pro Ile Glu Ala Met Ser Gly Gly Ser TrpIle Glu Thr Phe 1 5 10 15 His His 23 9 PRT Chimpanzee Antibody 23 IleLeu Met Phe Gly Val Leu Asn Ser 1 5 24 8 PRT Chimpanzee Antibody 24 AspArg Arg Gly Asp Phe Asp Phe 1 5 25 13 PRT Chimpanzee Antibody 25 Gly SerAsn Trp Asn Ser Phe Tyr Tyr Tyr Met Asp Val 1 5 10 26 8 PRT ChimpanzeeAntibody 26 Glu Gln Trp Arg Leu Tyr Asp Ser 1 5 27 18 PRT ChimpanzeeAntibody 27 Asp Arg Glu Val Tyr Pro Trp Asp Thr Tyr Phe Lys Pro Ser TyrPhe 1 5 10 15 Asp Phe 28 8 PRT Chimpanzee Antibody 28 Ser Gln Trp ArgAla Leu Asp Leu 1 5 29 8 PRT Chimpanzee Antibody 29 Asp Arg Arg Trp GluLeu Glu Ile 1 5 30 12 PRT Chimpanzee Antibody 30 Gly Asp His Thr Gly TyrVal His Tyr Phe Asp Ser 1 5 10 31 9 PRT Chimpanzee Antibody 31 Val ThrMet Val Gly Val Leu Thr Asp 1 5 32 14 PRT Chimpanzee Antibody 32 Glu GlyCys Ser Gly Leu Ser Cys Tyr Gly Ser Phe Asp Arg 1 5 10 33 30 DNAARTIFICIAL SEQUENCE Sequencing Primer 33 gcatgtacta gttgtgtcacaagatttggg 30 34 21 DNA ARTIFICIAL SEQUENCE Sequencing Primer 34attgcctacg gcagccgctg g 21 35 21 DNA ARTIFICIAL SEQUENCE SequencingPrimer 35 ggaagtagtc cttgaccagg c 21 36 21 DNA ARTIFICIAL SEQUENCESequencing Primer 36 acagctatcg cgattgcagt g 21 37 21 DNA ARTIFICIALSEQUENCE Sequencing Primer 37 cacctgatcc tcagatggcg g 21 38 28 DNAARTIFICIAL SEQUENCE Amplification Primer 38 atggcggtcg ctccggcccatgacaccc 28 39 33 DNA ARTIFICIAL SEQUENCE Amplification Primer 39ctattaaatg gagatagcgt agccaccaac agc 33 40 33 DNA ARTIFICIAL SEQUENCEAmplification Primer 40 ctattagcgg aactcaagtt cgagggcaaa gtc 33 41 33DNA ARTIFICIAL SEQUENCE Amplification Primer 41 ctattaagtc ggctcgccattggctgagac gac 33 42 33 DNA ARTIFICIAL SEQUENCE Amplification Primer 42ctattactgc gcgccggtcg caacattaac caa 33 43 31 DNA ARTIFICIAL SEQUENCEAmplification Primer 43 ctattaccga tgcccagcgg cattctcaac g 31 44 31 DNAARTIFICIAL SEQUENCE Amplification Primer 44 ctattatagc acagagtggggggctaaaac a 31

We claim:
 1. A polypeptide of about 30 amino acids in length, spanningfrom amino acids 572 to 607 of the open-reading frame 2 gene of ahepatitis E virus, wherein the polypeptide is a neutralization site forhepatitis E virus.
 2. A fragment of the polypeptide of claim 1, whereinthe fragment is a neutralization epitope for hepatitis E virus containedwithin the neutralization site according to claim
 1. 3. A nucleic acidmolecule encoding the polypeptide of claim
 1. 4. A nucleic acid moleculeencoding the fragment of claim
 2. 5. A DNA construct comprising thenucleic acid molecule of claim
 3. 6. A DNA construct comprising thenucleic acid molecule of claim
 4. 7. A method for producing neutralizingantibodies to hepatitis E virus, said method comprising administering toa mammal a pharmaceutical composition comprising the polypeptide ofclaim 1 in a manner sufficient to elicit production of said antibodies.8. A method for producing neutralizing antibodies to hepatitis E virus,said method comprising administering to a mammal an amount of thefragment of claim 2 coupled with a carrier protein sufficient to elicitproduction of said antibodies.
 9. A method for producing neutralizingantibodies to hepatitis E virus, said method comprising administering toa mammal an amount of the DNA construct of claims 5 or 6 sufficient toelicit production of said antibodies.
 10. A method of screening anantibody to hepatitis E virus to identify neutralizing antibodycomprising: a) incubating the antibody with the fragment of claim 1 or 2under conditions suitable to form a immune complex between the antibodyand the fragment; b) detecting the presence of said immune complex. 11.A neutralizing antibody to hepatitis E virus made by the method ofclaims 7, 8 or
 9. 12. A neutralizing antibody reactive with thepolypeptide of claim
 1. 13. A neutralizing antibody reactive with thefragment of claim
 2. 14. A method of detecting hepatitis E virus in abiological sample, comprising: (a) contacting the sample with at leastone antibody according to claims 11, 12 or 13 under conditions suitableto form an immune complex between the antibody and a hepatitis E virusantigen; and (b) detecting the presence of said immune complex.
 15. Themethod of claim 14, wherein the biological sample is selected from thegroup consisting of serum, saliva, plasma, bile, feces, lymphocytes,hepatocytes or other cells.
 16. A pharmaceutical composition comprisingthe polypeptide of claim
 1. 17. A pharmaceutical composition comprisingthe fragment of claim
 2. 18. A pharmaceutical composition comprising theDNA constructs of claims 5 or
 6. 19. A pharmaceutical compositioncomprising the neutralizing antibody of claims 11, 12 or
 13. 20. Avaccine for immunizing a mammal against hepatitis E infection, saidvaccine comprising the polypeptide of claim 1 coupled to a carrierprotein.
 21. A vaccine for immunizing a mammal against hepatitis Einfection, said vaccine comprising the fragment of claim 2 coupled to acarrier protein.
 22. A vaccine for immunizing a mammal against hepatitisE infection, said vaccine comprising a DNA construct of claim 5 or 6 ina pharmaceutically acceptable carrier.
 23. A method of providing passiveimmunoprophylaxis to a mammal or passive immunotherapy to a mammalinfected with hepatitis E virus comprising administering to said mammala therapeutically effective amount of a neutralizing antibody accordingto claims 11, 12 or
 13. 24. A monoclonal antibody that is immunoreactivewith the neutralization site of claim 1, said antibody having heavy (H)and light (L) chain immunoglobulin variable region amino acid sequencesselected from the group consisting of SEQ ID NOS: 1-4.
 25. A monoclonalantibody according to claim 24, wherein the heavy (H) and light (L)chain immunoglobulin variable region amino acid sequences are encoded bynucleic acid sequence selected from the group consisting of SEQ ID NOS:5-8.
 26. A method of detecting HEV in a biological sample, comprising:(a) contacting the sample with at least one monoclonal antibodyaccording to claim 24 under conditions suitable to form a complexbetween the antibody and a HEV antigen; and (b) detecting the presenceof said immune complex.
 27. The method of claim 26, wherein thebiological sample is selected from the group consisting of serum,saliva, plasma, bile, feces, lymphocytes, hepatocytes or other cells.28. A pharmaceutical composition comprising the monoclonal antibody ofclaim
 24. 29. A method of providing passive immunoprophylaxis to amammal or passive immunotherapy to a mammal infected with hepatitis Evirus comprising administering to said mammal a therapeuticallyeffective amount of a monoclonal antibody according to claim
 24. 30. Amonoclonal antibody having CDR3 sequence selected from the CDR3sequences shown in FIG. 8.